The extracellular matrix (ECM) has such profound effects on normal growth and differentiation that we have initiated a program to investigate its potential role in cancer, a disease involving abnormal growth and development. To study the interaction between a cell and its ECM as a function of oncogenic transformation we have used cultured chicken embryo fibroblasts infected with temperature-sensitive (ts) mutants of Rous sarcoma virus. We have discovered a novel protein (M about 21,000) in substratum-attached material (SAM), a specialized compartment of the ECM remaining attached to culture dishes after removal of cells with EGTA. The synthesis and deposition of the 21K protein in SAM are stimulated significantly, though transiently, during the early stages of transformation with ts mutants. Furthermore, increased amounts of the protein are not found in fully transformed cells infected with the wild-type virus. The protein is also induced by treatment of normal cells with a potent tumor promoter. We propose that the 21K protein may play an important role in the development of the transformed state, but not necessarily in its maintenance. The amphiphilic properties of this protein suggest a potential function in cross-linking and possibly organizing matrix components. ?The 21K protein does, in fact, bind to hyaluronic acid. Furthermore, it appears to be related to another 21K protein detected in the media of transforming cells?. We propose to isolate, purify and characterize the SAM 21K protein using chromatographic and immunological techniques. ?Monoclonal? antibodies to the protein will be ?produced by in vitro immunization? to aid in its characterization and purification and to describe its distribution in the matrix by immunofluorescence microscopy. Physical and chemical characterization and the potential interaction of the 21K protein with other matrix components will be established by electrophoretic, autoradiographic and binding studies. Finally, the potential role of the 21K protein in the expression of the transformed phenotype will be investigated by its ability to induce established transformation parameters in normal cells and ts virus-infected cells at the nonpermissive temperature.